Throughout this application various publications are referred to in brackets. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Glycans (oligosaccharides) are critical information carriers in biology, but progress toward understanding their roles has been hampered by lack of reagents that can detect subtle variations in glycan composition [1-4]. Antibodies and glycan-binding proteins (e.g., lectins) that recognize specific terminal sugars on oligosaccharides exist and are widely used, but these reagents have low affinity and are unable to distinguish among branched oligosaccharides [1, 2, 5]. Subtle changes in the glycan composition of cellular surface receptors, which can only be detected by discrimination of chemically similar high molecular weight branched glycans, are thought to signal major cellular events [1-4]. Therefore, reagents that can distinguish branched oligosaccharides from one another would be of high value in glycobiology research [5-12]. Antibodies with these capabilities are difficult to obtain using hybridoma methods because glycans themselves tend to be poorly immunogenic, and it is difficult to target antibody response to regions of oligosaccharides that would allow the desired differentiation [5, 8, 13, 14]. Furthermore, the molecular basis for selective and high-affinity glycan recognition by antibodies is poorly understood—there is much less structural data available for glycan-targeting antibodies than for antibodies that bind proteins [6, 7, 15-17]. Since glycans have much less hydrophobic functionality than do proteins and nucleic acids, glycan-protein interactions tend to be lower affinity than protein-protein or protein-nucleic acid interactions. Changes in cell surface glycan composition are associated with cancer and other disease states; therefore, high-affinity antibodies that target glycans have greater potential as diagnostic and therapeutic delivery agents. In particular, the tetrasaccharide Lewis Y antigen is highly expressed in cancer tissues and a LeY antibody-drug conjugate has been advanced through clinical trials [10]. High-affinity glycan antibodies would be important for deciphering glycan function in cancer and other diseases, and would provide a new avenue for the development of diagnostic and therapeutic delivery reagents.
The present invention addresses the need for methods that can be used to identify high-affinity antibodies that target glycans.